Aminoglycoside nephrotoxicity and renal function status.
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Aminoglycosides↗
Biomedical subjects
B L Bottemiller
Publications and source records attributed to B L Bottemiller.
Parathyroid hormone stimulates bone formation and resorption in organ culture: evidence for a coupling mechanism.
We have developed an in vitro system, using embryonic chicken tibiae grown in a serum-free medium, which exhibits simultaneous bone formation and resorption. Tibiae from 8-day embryos increased in mean (+/- SD) length (4.0 +/- 0.4 to 11.0 +/- 0.3 mm) and dry weight (0.30 +/- 0.04 to 0.84 +/- 0.04 mg) during 12 days in vitro. There was increased incorporation of [3H]proline into hydroxyproline (120 +/- 20 to 340 +/- 20 cpm/mg of bone per 24 hr) as a measure of collagen synthesis, as well as a 62 +/- 5% increase in total calcium and 45Ca taken up as an indication of active mineralization. A physiologic concentration (1 pM) of parathyroid hormone was found to stimulate bone resorption over control levels in this system. Parathyroid hormone stimulated the release of [3H]hydroxyproline from the bone shafts but not from the cartilage ends, indicating the specificity of the response. With 1 pM parathyroid hormone we observed an acute inhibition of bone formation, followed (after 12-16 hr) by a chronic stimulation of bone formation during the 12-day incubation. Both mineral uptake and matrix formation were enhanced at approximately the same rate during the 12-day incubation. The chronic enhancement of formation required parathyroid hormone only for the initial 8-10 hr of incubation. These results could be explained by the production or release of a factor from bone to stimulate formation in response to the acute increase in resorption--a "coupling factor." Indeed, dialyzed culture medium conditioned by actively resorbing bones stimulated bone formation over controls when added to organ cultures at a 1:20 dilution. The factor is larger than 12,000 daltons as determined by dialysis. The factor is specific for the bone shaft and did not affect the cartilage ends.
Animals↗
In vitro metabolism of 25-hydroxyvitamin D3 by isolated rat kidney cells.
Cells were dispersed from rat kidney after enzymatic digestion of the extracellular matrix. When the cells were suspended in a serum-free medium and incubated with (3)H-labeled 25-hydroxyvitamin D(3) (25-OH-D(3)) several polar metabolites, including 1,25-(OH)(2)[(3)H]D(3) and 24,25-(OH)(2)[(3)H]D(3) were produced. The specific activities of the 25-OH-D(3):1- and 24-hydroxylases in isolated rat kidney cells were 10-100 times greater than in avian kidney homogenates. The rates of production of 1,25-(OH)(2)D(3) and 24,25-(OH)(2)D(3) were linear over a wide range in cell densities (0.65-5.0 x 10(6) cells per ml) and substrate concentrations (3.5-70 nM). The rate of production of 24,25-(OH)(2)[(3)H]D(3) from 25-OH-[(3)H]D(3) by cells isolated from rats fed control diet was linear with time for up to 30 min, while the synthesis of 1,25-(OH)(2)[(3)H]D(3) was linear for over 90 min. The specific activity of the 25-OH-D(3):1-hydroxylase was increased in kidney cells from vitamin D-deficient rats (11.5 fmol/min per 10(6) cells) as well as calcium-deficient rats (8.1 fmol/min per 10(6) cells) when compared to cells from rats fed the control diet (2.0 fmol/min per 10(6) cells). Also, the specific activity of the 25-OH-D(3):24-hydroxylase was reduced in cells from the vitamin D-deficient rats (<0.2 fmol/min per 10(6) cells) and calcium-deficient rats (5.1 fmol/min per 10(6) cells) compared to the controls (15.2 fmol/min per 10(6) cells). On the basis of these results, as well as previous in vivo studies, we conclude that the metabolism of 25-OH-D(3) by freshly isolated rat kidney cells reflects the in vivo activities of the renal vitamin D-metabolizing enzymes and may prove useful as an assay.
25-Hydroxyvitamin D3 1-alpha-Hydroxylase↗
Serum-free culture of Japanese quail kidney cells. Regulation of vitamin D metabolism.
Cells obtained from male quail kidneys by digestion with collagenase and hyaluronidase were plated and maintained in a chemically defined, serum-free medium. Culture dishes (35 mm) were inoculated with 1.5 . 10(6) cells which became confluent in 5 days. The cells maintained an epithelial-like morphology over the entire culture period. During a 2 h incubation the cells metabolized 25--30% of the 10 nM 25-hydroxyvitamin D-3 (25-OH-D-3) provided. Seven metabolites were chromatographically separated on Sephadex LH-20. Three have been identified as 1 alpha, 25-dihydroxyvitamin D-3 (1,25(OH)2D-3), 24,25-dihydroxyvitamin D-3 (24,25(OH)2D-3) and 1 alpha, 24,25-trihhydroxyvitamin D-3 (1,24,25(OH)3D-3). The activities of the 25-OH-D-3:1 alpha- and 24-hydroxylases increased eight times faster than the cell number in 5 days. Preincubation of the cells with 10 nM 25-OH-D-3 or 1,25(OH)2D-3 decreased 1,25(OH)2D-3 synthesis, and increased both 24,25(OH)2D-3 and metabolite IV synthesis. The decrease in 25-OH-D-3:1 alpha-hydroxylase activity required a 2 h preincubation with 25-OH-D-3, while stimulation of 25-OH-D-3:24-hydroxylase activity and metabolite IV production required a 6 h preincubation. Incubations of cells for 1 h with parathyroid hormone resulted in a 30-fold increase in cyclic AMP in the medium. A 6 h preincubation with parathyroid hormone decreased 24,25(OH)2D-3) synthesis 50% relative to control cells. These results demonstrate the amenability of this system for studying the regulation of 25-OH-D-3 metabolism, as well as its use for other in vitro studies on renal cell function in a chemically defined culture system.
Animals↗